JP2007527713A - Use of indanoylamide to stimulate secondary metabolism in yew species (Taxussp.) - Google Patents

Abstract

  The present invention is directed to a method for producing taxanes by culturing yew suspension cells in a nutrient medium containing indanoyl amino acids. Indanoyl amino acids may be added batchwise at any time during the culture or in the feed stream. In particular, the synthetic compounds 6-ethyl-indanoyl-isoleucine, 6-bromoindanoyl isoleucine, and 1-oxo-indan-carboxy- (L) -isoleucine-methyl ester amide (1-OII) are used in the yew cell culture. Has been found to increase taxane production from

Description

  The present invention relates to the use of indanoylamide and indanoyl amino acid conjugates such as coronolone, and methods for accumulating taxanes in plant cell cultures of taxane-producing species useful for the production of pharmaceutical compositions, and It relates to a treatment method.

  Analogs such as paclitaxel and docetaxel are at the forefront of development in cancer treatment and there is a growing global demand for these products. Because the total chemical synthesis of paclitaxel is complex and uneconomical, paclitaxel and its analogs are currently produced commercially by semi-synthetic or biosynthetic methods. Semi-synthesis requires the use of natural, advanced taxane precursors (which are closely related to paclitaxel structure) and complex to complete the structural conversion of these precursors to paclitaxel or related taxanes. With chemical reaction. These precursors include 10-deacetylbaccatin III, baccatin III, N-acylated taxanes and the like. Various patents and papers disclose the chemical conversion of diterpenoid cores into biosynthetic pathways, biosynthetic genes, enzymes or desired taxanes. For specific examples of these disclosures, see Patent Document 1, Patent Document 2, Patent Document 3, Patent Document 4, Patent Document 5, Non-Patent Document 1, and Non-Patent Document 2.

  The biosynthesis of paclitaxel is achieved through the use of yew species plant cell cultures. The advantage of a biosynthetic pathway is that the desired compound or composition can be produced directly in a relatively pure form without the need for additional chemical reactions. Several publications disclose taxane production methods by biosynthetic methods. Patent Document 6 (Christen et al., 1991) describes the production and recovery of paclitaxel and taxane-like compounds by cell culture of Taxus brevifolia. U.S. Patent Nos. 5,099,047 (Bringi et al., 1993) and U.S. Patent No. 5,047,059 (Bringi et al., 1995) describe improved production and recovery of paclitaxel and other taxanes by cell culture of yew species. Bringi et al. Earliest disclose a profitable plant cell culture process for taxane production.

Patent Document 9 (Choi et al., 2001) describes a method for producing paclitaxel in a medium in the process of semi-continuous culture of yew cells by adding sugar or AgNO ₃ together with sugar. Other related patents include, for example, Patent Document 10 and Patent Document 11.

  Patent Document 12 (Yukimune, 1995), Patent Document 13 (Yukimune et al.), And Patent Document 14 (Bringi et al., 1997) include processes such as heavy metal compounds, heavy metal complex ions, heavy metal ions, amines, or anti-ethylene agents. The production of taxanes by culturing tissue or cells in the presence of an agent that enhances and under controlled oxygen concentrations is described. These patent applications also disclose the beneficial effects of jasmonic acid and related compounds to increase taxane yield in yew cell cultures. Compounds that enhance these patent-pending processes are free of aromatic rings.

  Compounds related to jasmonic acid and their uses are described in, for example, Non-Patent Document 3, Non-Patent Document 4, Non-Patent Document 5, Non-Patent Document 6, Yamada et al., EP 86102811, “Process for Production of Oxime Derivatives”. 6 of Patent Document 15 and Non-Patent Document 7.

Haider et al. Teach that analogs of jasmonic acid induce mechanotransduction but in many cases do not induce the production of secondary metabolites. Furthermore, Non-Patent Document 1 (p. 744, second column, first paragraph) also reported that when coronatine is aromatized, it becomes planar and loses its biological effect. Haider et al. Point out the following:
Activation of defense genes and activation of genes for mechanotransduction appears to be regulated by various compounds present in the process of the jasmonic acid biosynthetic pathway. Coronatine, a closed structure analog of 12-oxo-phytodienoic acid, and octadecanoids are powerful inducers of tendril wrap. On the other hand, methyl jasmonate is much more effective than octadecanoids or coronatine in activating benzo [c] phenanthridine alkaloid accumulation. These results suggest that various physiological responses, such as those against herbivory or pathogens, especially mechanotransduction, are activated by the octadecanoid pathway and can be separated by chemical derivatization of inducer molecules. Add support (Blechert et al., 1997). For example, protective gene activity in this paper using [three analogs of jasmonic acid], all of which are potent inducers of benzo [c] phenanthridine alkaloid accumulation in E. californica This hypothesis is strongly due to the various results obtained with regard to crystallization and the failure of the same compounds by Blechert et al. (1999) to induce mechanotransduction in Bronia. Proven.

  Boland et al. Describe aspects related to the physiology of plants exposed to jasmonic acid and / or coronatine. For example, see Non-Patent Document 8, Non-Patent Document 9, Non-Patent Document 10, Non-Patent Document 11, Schuler et al., Patent Document 16 entitled “6-Substituted Indanoyl Amino Acid Conjugates as Mimics to the Biological Activity of Coronatine”. Let ’s do it.

  Patent Document 16 describes a 6-position substituted indanoyl amino acid as a plant regulator. Schuler et al. (US Pat. No. 6,057,099) described that “6-ethyl-1-oxo-indanoylisoleucine methyl ester is a potent inducer of the winding response of a tendril that is sensitive to the contact of Brionia diocia. It is pointed out. However, Patent Document 16 did not disclose the use of cell culture and yew species for improving taxane production.

  Thus, in view of the teachings of U.S. Patent No. 6,099,059 (Yukimune et al.), Where coronatine has been shown to induce paclitaxel and taxane biosynthesis, planar aromatized analogs of coronatine are biologically biocompatible for this purpose. It is expected that it will not be effective.

  Any of these plant cell culture-based methods can be used, for example, for 6-ethyl-indanoyl-isoleucine (6) for improving or modifying the taxane production of suspension culture cells, and more particularly yew cells. It is important not to disclose the use of -EII). Given the observations of Schuleer et al. (US Pat. No. 6,057,049) that indanoyl amino acid conjugates were a potent inducer of wrapping wrapping (mechanotransduction), these specific analogs were found in yew cell cultures. It is surprising to induce the production of taxane secondary metabolites from. In addition, Haider et al. Suggest that aromatization that changes coronatine analogs to planarity also turns them biologically inactive. However, 6-EII (having an aromatic ring) is both planar and active. Thus, it is even more surprising that planar coronatine analog indanoyl amino acid conjugates are effective inducers of taxane production in yew cell cultures.

US Pat. No. 5,994,114 US Pat. No. 5,200,2544 US Pat. No. 6,437,154 US Pat. No. 6,287,835 US Pat. No. 6,265,639 US Pat. No. 5,019,504 International Publication No. 93/171112 US Pat. No. 5,407,816 US Pat. No. 6,248,572 US Pat. No. 5,665,576 US Pat. No. 6,429,889 European Patent Application No. 0683232 European Patent Application No. 0727492 WO97 / 44476 pamphlet European Patent No. 0194554 International Publication No. 02/055480 Pamphlet US Pat. No. 6,452,024 US Pat. No. 6215000 specification US Pat. No. 6,136,989 US Pat. No. 6,124,482 US Pat. No. 5,281,727 US Pat. No. 5,380,916 US Pat. No. 5,969,165 US Pat. No. 5,300,377 US Pat. No. 5,393,896 US Pat. No. 5,393,895 US Pat. No. 5,549,830 US Pat. No. 5,654,448 US Pat. No. 5,723,635 US Pat. No. 5,736,366 US Pat. No. 5,744,333 US Pat. No. 5,756,098 US Pat. No. 6,800,485 "Synthetic Routes to the Diterpenoid Cores of Phorbol and Resiniferatoxin," Eckelbarger, J.D. 2001 "Taxol biosynthetic genes," Walker, K. et al., Phytochemistiy 58 (2001) 1-7 Haider, et al., "Structure-Activity Relationships of Synthetic Analogs of Jasmonic Acid and Coronatine on Induction of Benzo [c] phenanthridine Alkaloid Accumulation in Eschscholzia californica Cell Cultures," Biol. Chem., Vol. 381, pp. 741-748 , August 2000 Krumm, et al., "Leucine and Isoleucine Conjugates of 1-Oxo-2,3-dihydro-indene-4-carboxylic Acid: Mimics of Jasmonate Type Signals and the Phytotoxin Coronatine," Molecules 1996, 1, 23-26 Miersch, et al., "Structure-activity Relations of Substituted, Deleted or Stereospecifically Altered Jasmonic Acid in Gene Expression of Barley Leaves," Phytochemistry 50 (1999) 353-361 Schuler, et al., "Coronalon: a Powerful Tool in Plant Stress Physiology" Boudier, et al., "Stereoselective Preparation and Reactions of Configurationally Defined Dialkylzinc Compounds," Chem. Eur. J. 2000, Vol. 6, No. 15, 2748-2761 Boland, et al., "Jasmonic acid and Coronatine Induce Odor production in plants", Angewandte Chemie-International Edition, 43: 1600-1602 (1995) Krumm, et al., "Induction of volatile biosynthesis in the Lima bean (Phaseolus lunatus) by lucine- and isoleucine conjugates of 1-oxo- and 1-hydroxyindan-4-carboxylic acid: Evidence for amino acid conjugates of jasmonic acid as intermediates in the octadecanoid signaling pathway ", FEBS Lett. 377: 523-529 (1995) Schuler, et al., "Synthesis of 6-azido-1-oxo-indan-4-oyl isoleucine; a photoaffinity approach to plant signaling", Tetrahedron, 55: 3897-3904 (1999) Lauchli, et al., "Indanoyl Amino Acid Conjugates: Tunable Elicitors of Plant Secondary Metabolism," The Chemical Record, Vol. 3, 12-21 (2003)

  One embodiment of the present invention is directed to a method for producing taxanes by culturing yew suspension cells in a nutrient medium comprising indanoyl amino acids. Another embodiment of the present invention is directed to a nutrient medium for plant cell culture comprising an indanoyl amino acid. The indanoyl compound may be added into the fed batch feed stream at any point during the culture. In a preferred embodiment, the nutrient medium during suspension culture is partially depleted of carbon before additional carbon source is included in the feed stream.

  In each embodiment of the present invention, multiple potentiators and / or inhibitors may be added to the nutrient medium. In a preferred embodiment, indanoyl amino acid is added in an amount effective to alter the profile of the taxane produced by the culture compared to the absence of indanoyl amino acid. In another embodiment, the indanoyl amino acid is provided in an amount effective to selectively increase baccatin III compared to the absence of indanoyl amino acid.

  In each of the listed embodiments, the indanoyl amino acid is selected from the compounds described in US Pat. More preferably, the indanoyl amino acid is an unsubstituted indanoyl amino acid (1-oxo type) or a 6-position substituted indanoyl isoleucine and derivatives thereof. Most preferably, the indanoyl amino acid is 6-ethylindanoylisoleucine (6-EII), 6-bromoindanoylisoleucine (6-BII), 1-oxo-indane-carboxy- (L) -isoleucine-methyl ester amide (1-OII), or a mixture thereof. It should be understood that any of the indanoyl amino acid compounds may be used alone to induce the production of the desired taxane.

  In each of the listed embodiments, a preferred method of increasing taxane production involves the use of additional enhancers, more preferably silver compounds and complexes, related compounds of methyl jasmonate, and phenylpropanoid inhibition. Selected from substances. Similarly, the production of preferred taxane products may be stimulated by feeding various biosynthetic precursors of these taxanes to the medium at the appropriate stage. Furthermore, it is possible to separate rapid biomass growth and rapid product production and that different formulations of nutrient media and different culture conditions may be used for the growth and production stages. Recognized in the technical field.

  In another embodiment, the nutrient medium comprises auxin, a compound having auxin-like growth regulatory activity, or a mixture thereof. In another embodiment, the nutrient medium comprises silver ions, silver compounds, silver complexes, or mixtures thereof. In another embodiment, the nutrient medium contains an inhibitor of phenylpropanoid metabolism. In a preferred embodiment, the inhibitor of phenylpropanoid metabolism is a compound having a methylenedioxy group, more preferably the inhibitor of phenylpropanoid metabolism is MDCA (3,4-methylenedioxycinnamic acid). Or related compounds such as methylenedioxynitrocinnamic acid, 3,4-methylenedioxyphenylacetic acid, methylenedioxyphenylpropionic acid. In another embodiment, the nutrient medium further comprises an amino acid, more preferably the amino acid is glutamine.

  In a preferred embodiment, the indanoyl amino acid is provided in an amount that is protective against silver toxicity.

  The present invention provides methods for increasing taxane accumulation in plant cell cultures of taxane producing species and methods for isolating and purifying them. The present invention relates to a specific enhancer (indanoylamide) used alone or in combination with other enhancers to improve the yield of taxanes as paclitaxel, baccatin III, and other taxane analogs. Is targeted.

  Each document described throughout this specification is hereby incorporated by reference in its entirety to the extent that it does not conflict with the disclosure of this specification. Specifically, these references include both growth and production media that yield high taxane yields from callus culture, including adding and manipulating culture conditions to improve yield. A preferred method of development is provided.

  Taxanes are a member of a group of compounds also known as taxoids or taxane diterpenoids. Taxanes are characterized by a tricyclic diterpene / taxane ring system. Currently, more than 300 taxanes are known. The terms “paclitaxel-like compound” or “taxane” are used interchangeably to describe a diterpenoid compound having a taxane ring. Taxanes can themselves have anti-tumor activity or can be modified to yield compounds with biological activity. Various specific taxanes are also described in US Pat. Nos. 6,099,086 (Bringi et al., 1997), US Pat.

  The term “taxane producing cell” means any cell capable of producing a taxane molecule under at least one set of culture conditions. The term “taxane-producing species” means any species capable of producing taxane molecules under at least one set of culture conditions. The term “taxane producing cell culture” means any culture comprising “taxane producing cells”. The biosynthetic tissue (ie, taxane producing cells) can be selected from any taxane producing species (or combination thereof), as is known to those skilled in the art. The yew species from which the tissue is selected are Taxus brevifolia, Taxus canadensis, Taxus cuspiddata, Taxus baccata, Taxus globusa, Taxus globosa Taxus floridana, Himalayan yew (Taxus wallichiana), Taxus media, Taxus chinensis, and Taxus gena are preferred. More preferably, the yew tissue is selected from T. chinensis. Alternatively, the tissue may be selected from one or more Torreya species. The Kaya species is preferably Torreya gladifolia or Torreya californica. Alternatively, the tissue may be selected from one or more Corylus species. The hazel species is preferably Corylus avella. The present invention contemplates the use of cell combinations from various species, variants, strains, and / or genera in any manner. For example, but not limited to, one or any combination of yew cells may be used in combination with one or any combination of kaya species. The use of tissues derived from hybrid plants, genetically modified plants, and the like is also contemplated.

  The term “callus” is used to describe a mass of cultured plant cells that are structurally undifferentiated and cultured on a solidified medium. The term “suspension culture” is used to describe structurally undifferentiated cells dispersed in a liquid nutrient medium. It will be appreciated that a suspension culture contains cells at various assembly stages. A range of aggregate sizes occurs in the suspension, ranging from tens of microns in diameter (single cells or several assembled cells) to aggregates of thousands of diameters consisting of thousands of cells. It reaches.

  The term “nutrient medium” is used to describe a medium suitable for cultivation of plant cell callus and suspension cultures. The term “nutrient medium” is common and encompasses both “growth medium” and “production medium”. The term “growth medium” is used to describe a nutrient medium that promotes rapid growth of cultured cells. The term “production medium” refers to a nutrient medium that promotes biosynthesis of paclitaxel, baccatin III, or total taxane in cultured cells. It will be appreciated that growth can occur in the production medium, production can occur in the growth medium, and both growth and production can occur in a single nutrient medium. Preferably, the growth and production stages of the taxane producing cell culture are distinguishable and the nutrient medium is independently optimized.

  If all nutrients are supplied first and the culture contents, including cells and products, are collected at the end of the culture phase, this mode of operation is referred to as a “one-stage batch process”. If the batch process is divided into two successive stages, the growth stage and the production stage, and the medium is changed between the two stages, this mode of operation is called “two-stage batch process” . Within the contemplation of the present invention, the transition from the growth medium to the production medium may take place by an abrupt step change, by a series of steps or by a progressive continuous change. In one extreme, gradual change is achieved by gradual replacement of medium that gradually changes composition. In another alternative, the gradual change is achieved by feeding one or more components of the production medium into the growth phase culture. This is an example of a fed-batch process. In a “fed-batch” operation, certain media components, such as nutrients and / or one or more enhancers, are supplied periodically or continuously during all or some period of one-stage or two-stage culture. The A description of nutrients and potentiating substances can be found in Table A or Tables 1 and 2 of US Pat. In addition, a combination of abrupt changes and incremental changes can be used. In one embodiment, some portions of the nutrient medium may be changed abruptly while other ingredients are being slowly fed.

  According to embodiments of the present invention, if a significant portion of the contents of a batch culture is recovered and fresh media for continuous cell growth and production is added, the method is , Similar to the “repeat draw and fill” operation, referred to as the “semi-continuous method”.

  If fresh media is continuously fed and the effluent media is removed continuously or repeatedly, according to an embodiment of the invention, the method is called “continuous”. According to another embodiment, when the cells are retained in the reactor, the method is called “perfusion”. If the cells are continuously removed along with the effluent medium, according to another embodiment of the invention, this continuous process is referred to as a “chemostat”.

Indanoylamides contemplated by the present invention as particularly useful potentiators for improving or modifying indanoylamide taxane production are described below. Compounds and methods of making these compounds are described in US Pat.

  Coronatine has the following formula:

  As described by Borand et al., Indanoylamide was considered as an analog of coronatine. Among the compounds described in Patent Document 16, this compound is preferably an indanoyl amino acid conjugate (also referred to as indanoyl amino acid). Preferably, the present invention contemplates the use of compounds that can be represented, for example, by Boland et al.

Boland describes various substituents of the R group, each intended for use with the present invention, although preferred R groups include the following:
R ₁ = double bonded oxygen,
R ₂ = lower alkyl group such as methyl group or ethyl group, or halogen, preferably bromide,
R ₃ = methyl or hydrogen,
R ₄ = Amino acid side chain. Preferably, the amino acid is a nonpolar natural or synthetic amino acid. More preferably, the amino acid is selected from glycine, valine, alanine, leucine, isoleucine, and proline. In a particularly preferred embodiment, the amino acid is isoleucine.

  According to the present invention, indanoylamide is used in place of or in addition to the inducers described below. Preferably, indanoyl derivatives and analogs are used in the range of 10-500 μmol / liter, more preferably 50-250 μmol / liter.

Cell culture methods and media: Manipulation of culture conditions and enhancers The culture conditions can be manipulated to promote the production of the desired taxane. For example, reaction conditions such as temperature, pH, darkness, removal or addition of nutrients or other agents, changes in nutrient or agent concentrations, or combinations thereof can be manipulated.

  These compositions may further comprise any of a number of additional ingredients, such as nutrients and enhancers. Preferably, the composition comprises, as discussed below, an inducer, a jasmonic acid related compound, a compound that affects ethylene biosynthesis or action, particularly an inhibitor, an inhibitor of phenylpropanoid metabolism, an anti-aging agent, Includes precursors and potentiators such as growth regulators associated with auxin.

  Prior to initiating cell culture, the tissue may be selected based on other parameters such as being able to promote the formation of a specific taxane or taxane precursor under specific conditions, or to promote formation ( For example, the tissue may be treated chemically, genetically, or otherwise.

  The production of secondary metabolites is a complex process that requires the concerted action of many different enzymes to generate and sequentially modify precursors that are ultimately converted to the desired secondary metabolite. At the same time, production of secondary metabolites may be reduced if other enzymes metabolize the desired metabolite precursors and deplete the precursor pool required to form secondary metabolites. .

  Taxanes are secondary metabolites that are produced through a series of numerous enzymatic steps, and several types of potentiators that enhance taxane biosynthesis are known (eg, Table A, US Pat. 1997), Patent Document 12 (Yukimune, 1995), Patent Document 13 (Yukimune et al.)). It is believed that the addition of one of these potentiators to the taxane producing cell culture increases the taxane production rate. Furthermore, the use of enhancers is believed to have at least some enhancing effect in many taxane production cultures, because the overall production rate is not between a single rate-limiting step, but between multiple rate-limiting factors. It is suggested that it depends on the complex interaction. Removing any one of the rate-limiting factors is thought to enhance taxane production, but the magnitude of the enhancement is relative to other stages of taxane biosynthesis after certain constraints are removed. It will vary depending on the individual culture conditions that determine the specific rate-limiting effect.

  Culture conditions that affect the interaction between various rate-limiting factors include the genetic makeup of the cells, the composition of the medium, and the gas environment, temperature, lighting, and process procedures. The enhancer added to a particular culture usually takes into account the rate-limiting factor in that culture, which can be determined experimentally by comparing the effects of the individual enhancers shown herein, Selected. Enhanced taxane production can be achieved when multiple enhancers are present in the culture. The types of enhancement substances are anti-browning substances, anti-aging substances, anti-ethylene agents, plant growth regulators, precursors, inhibitors, inducers, and stimulants. Exemplary enhancers and general requirements are described, for example, in the references cited in the background section above. In a preferred embodiment, the medium can be specifically adjusted to promote growth or taxane production. Based on the guidance provided herein and in view of the generally understood principles of plant cell physiology and metabolism, one of ordinary skill in the art can easily develop a medium to promote growth or paclitaxel / taxane production. It can be developed on its own.

  Methods for callus formation and callus growth of taxane producing tissues are known in the art, along with preferred means for altering growth morphology, productivity, product profile, and other characteristics. After the callus culture is constructed, the cells are then cultured in production medium and / or growth medium. In particular, mass production of taxanes is facilitated by culturing taxane producing cells in suspension culture. In general, suspension culture can be initiated using a medium that has been successfully cultured in callus. However, the requirements for suspension culture, in particular for the highly efficient production of taxanes, can be better met by medium improvements. It has been found that culturing taxane-producing cells in improved media and processing parameters substantially increases the yield of one or more taxanes from the culture. Suspension cultures that produce taxanes can achieve rapid growth rates and high cell densities when appropriate nutrients and reaction conditions are used. Based on this specification and the guidance provided in the patent, which is incorporated by reference in its entirety, to achieve optimal results, certain types of ingredients and ingredients derived from those within a given class Incorporating, modifying, and manipulating is normal for those skilled in the art.

  The present invention contemplates the use of an improved biosynthetic process to obtain the desired result. According to an embodiment of the present invention, the taxane-producing cells are cultured under conditions capable of producing a taxane. These include conditions that promote biomass accumulation and / or taxane production. The culture of taxane producing cells is described in detail in, for example, Patent Document 8 (Bringi et al., 1995) and Patent Document 14, and the improvement of these taxane production conditions is described below, and Example 1 below is described. To 8. However, the present invention is not limited to them. One skilled in the art can prepare suitable taxane producing cell cultures according to the guidance provided herein with references incorporated by reference.

  Various culture conditions and enhancers are known in the art, such as in the references described throughout this application, but preferred conditions are described below.

Preferred cell culture embodiments Cell culture initiation includes, for example, careful washing with clean water, use of bactericides such as hypochlorite, use of wetting agents such as Tween and Triton, use of antibiotics, And optionally sterilizing the surface of plant source materials, such as the use of antifungal agents. Then, the plant part may be used as it is, or a part thereof such as an embryo taken out from the seed may be used. Next, standard nutrient media suitable for the formation of yew callus, temperature range and pH range are used as culture conditions. Similarly, gelling agents, reduced pigmentation, activated carbon, etc. and standard light-dark cycles are used.

  Callus growth is the development of a substantially undifferentiated cell mass attached to a plant part that is carefully removed and grown as an undifferentiated culture. Culture conditions relating to preferred media, pH ranges, preferred carbon sources, nitrogen sources, macro and micro salts, vitamins, and growth regulators are all described, for example, in US Pat. In addition to the procedures described in those documents, preferred gelling agents include agar, hydrogel, gelatin, and gelrite. Similarly, charcoal is preferably used to remove unwanted by-products and undesirable organic compounds. Inoculum is usually in the range of about 0.01 to 10 g / 25 ml. In addition, subculture techniques are preferably utilized to periodically transfer multiple callus portions into a fresh nutrient source.

  The conditions for suspension culture are described in Patent Document 14, for example. In addition to the procedures described in that document, after initiation, substantially separate the cells from the medium (usually by filtration) and then reintroduce a portion into the nutrient-containing medium, or a volume of Suspension culture by transferring the culture broth (cells and medium) to nutrient-containing medium or by allowing the cells to settle and then removing any portion of the already existing medium and reintroducing the nutrient-containing medium The product may be further cultured. When cells are separated and transferred to another nutrient-containing medium, the amount transferred may range from 0.3% to 30% based on the wet weight, but from 1% to 25% of the wet weight. % Is preferred. Note that this ratio can vary as cells acclimate and / or grow. When transferring cells and media by volumetric measurement, the ratio of the transferred volume to the final volume may be from 1% to almost all of the volume. In this case, fresh nutrients may be supplied in concentrated form so that only a small volume increase occurs. In this way, the culture can be divided into a plurality of parts. Each part can optionally be used for taxane production. The composition of the nutrient-containing medium need not be the same for their various parts. Other components not included in the original medium may be added, or items derived from the original medium may be omitted or their concentrations may be changed. The culture period is usually at least 2 days. In addition, the growth period may be extended by supplementing the partially depleted medium with additional nutrients.

  All concentrations refer to the average initial value in the extracellular medium. The concentration in the feed solution and thereby the concentration in contact with the cells can be higher than the concentration indicated locally. In addition to nutrients commonly used in plant cell culture, other components may be added to support taxane production. Ingredients that are particularly suitable for taxane production include one or more selected from inducers, stimulants, precursors, inhibitors, growth regulators, heavy metals, and / or ethylene inhibitory compounds. Inducers include jasmonic acid and related compounds, tuberonic acid and related compounds, cucurbic acid and related compounds, coronatine and related compounds, 6-ethyl-indanoylisoleucine and related compounds, 12-oxo-phytodienoic acid and related compounds, cystemin And related compounds, borititin and related compounds, oligosaccharides, chitosan, chitin, glucan, cyclic polysaccharides, bacteria, fungi, yeast, plants, insects, or cellular substances derived from substances contained in insect saliva or secretions Preparations containing, inhibitors of ethylene biosynthesis or action in plants, in particular compounds or complexes containing silver, cobalt, aminoethoxyvinyl glycine, phenylalanine ammonia lyase, cinnamic acid hydroxylase, coumarate CoA liger Inhibitors of phenylpropanoid metabolism such as compounds known to inhibit methylenedioxycinnamic acid, methylenedioxynitrocinnamic acid, methylenedioxyphenylpropionic acid, generally methylenedioxyphenylacetic acid, methylene Other compounds containing a methylenedioxy functional group such as dioxybenzoic acid. Non-limiting examples of growth regulators and / or inhibitors and combinations thereof include, but are not limited to, the tables disclosed in Table A and US Pat. Amino acids include any common amino acid utilized in cell culture, such as glutamine, glutamic acid, aspartic acid, α- or β-phenylalanine.

  Inducing agents such as jasmonic acid related compounds may generally be used at doses ranging from 0.01 μmol / liter to 1 mmol / liter. Preferably, this value will be between 1 and 500 μmol / liter. Preparations containing cellular material may be added based on the concentration of the individual components of the preparation or as some fraction of the culture volume. Heavy metals such as silver salts or complexes and ethylene inhibitors may be used at concentrations up to 1 mmol / liter, but usually the range is 0.01-500 μmol / liter. Other inhibitors may be used at a concentration of 1 μmol / liter to 5 mmol / liter. Aromatic compounds containing methylenedioxy functional groups may be included at a concentration of 0.1 μmol / liter to 5 mmol / liter, but more typically from 1 μmol / liter to 2 mmol / liter. Growth regulators may be used at values ranging from 0.001 μmol / liter to 2 mmol / liter, but more commonly they are used at concentrations ranging from 0.01 μmol / liter to 1 mmol / liter. be able to. Precursors such as amino acid or terpenoid precursors may be used at concentrations in the range of 1 μmol / liter to 20 mmol / liter, but more commonly they are used at concentrations in the range of 10 μmol / liter to 10 mmol / liter. use. By following the guidance provided herein, one of ordinary skill in the art will be able to find individual advantages of using materials alone or in combination by routine experimentation outside the indicated useful range. These findings are considered to be within the scope of the present invention.

  The components that supply the cells can be provided in a number of different ways. Components may be added to specific growth stages such as lag phase, log phase, or stationary phase. All ingredients may be fed simultaneously and then the taxane may be recovered after an appropriate time. Alternatively, not all the components need to be provided at the same time. Conversely, one or more of them may be supplied at different times during culture. Furthermore, the addition may be performed discontinuously with respect to the initial contact, or at a time offset, and the duration of such feeding may vary depending on various components. You may supply a component to several parts. The taxane can then be recovered. One or more components may be provided as part of a solution that is separately contacted with the cell culture or a portion thereof. Taxanes can be recovered from the entire culture or a portion of the culture (medium only, cells only, or some amount of cells and medium). The taxane can be recovered at any time during the culture or after the completion of the culture period. A portion of the culture can be removed at any time or periodically and used for taxane production and / or recovery, or for further growth of cells. If desired, such cell-containing portions may be further exposed to nutrients or other ingredients.

  In one embodiment, media containing nutrients or other ingredients may be added to replenish some or all of the removed volume. The replenishment (dilution) rate (volume addition rate divided by the volume of liquid in the container) may vary between 0.1 and 10 times the individual growth rate of the cells. A portion of such removed material may be added back into the original culture, for example, cells and media may be removed and those media or cells may be used for taxane recovery and the remaining cells Alternatively, the medium may be returned. The feed rates of the components to the culture, and the levels of the various components in the culture, can be adjusted so that the taxane can be advantageously produced and recovered. Separate portions of the culture may be exposed to the components in any of the previously described embodiments and then mixed at a ratio determined to favor taxane production. The cell content of the culture can also be adjusted to advantageously produce taxanes or grow cells. Adjustment of the cell content can advantageously be used in conjunction with methods for contacting nutrients or other ingredients.

The levels of gases such as oxygen, carbon dioxide and ethylene can be adjusted to advantageously produce one or more taxanes or to promote biomass accumulation. Routine cultures in laboratory scale culture vessels performed in an air atmosphere using normal closures such as sheets, plugs, or caps have dissolved oxygen levels below air saturation, as well as in the atmosphere. Results in higher CO ₂ and ethylene levels than are present in Thus, typically, the headspace carbon dioxide level of the culture is generally higher than about 0.03%. However, the concentration of carbon dioxide and / or ethylene can advantageously be adjusted to produce taxanes or grow cells. That is, the inventors have found that for better taxane production, this CO ₂ level is higher than 0.1% (about 3 times the atmosphere) up to 10%, preferably between 0.3 and 7%. It was found that it is preferable. Ethylene may preferably be less than 100 ppm, preferably less than 20 ppm as measured in the gas phase in equilibrium with the liquid phase at that temperature. Adjusting the dissolved gas by one or more methods including changing the agitation rate, composition of the aeration gas, supply or exhaust rate of the aeration gas, or by adjusting the total pressure of the culture vessel it can.

Each gas may be supplied independently. For example, the source of oxygen and CO ₂ may be different. The stirring speed may be adjusted to 1 to 500 times per minute (by rotation or vibration of the stirring group or fluid circulation). The gas supply rate may be 0.01 to 10 volumes of gas per unit volume of culture broth per minute. Even if it is supplied directly to the culture solution, it is supplied to the separated liquid portion, and then the portion is cultured. May be mixed with the rest of the culture or fed into the headspace of the culture. In general, the dissolved oxygen concentration advantageous for taxane production and yew cell growth can be adjusted to 10-200% of air saturation at working temperature, preferably 20-150% of air saturation. Of course, due to various operational reasons, such as a temporary reduction in aeration, the dissolved oxygen level may be as low as zero for periods ranging from minutes to hours. Individual useful combinations of oxygen, carbon dioxide, and ethylene outside these ranges can be found by routine experimentation and are considered within the scope of the present invention.

  In one embodiment, the media can be specifically adjusted to promote growth or taxane production. Based on the guidance provided above and based on generally understood principles of plant cell physiology and metabolism, one skilled in the art can devise a medium to promote growth or paclitaxel / taxane production. I will. For example, the temperature range may be changed (for example, 0 to 35 ° C, preferably 15 to 35 ° C, more preferably 20 to 30 ° C). Similarly, the period of the light / dark cycle may be changed.

  In a preferred embodiment, the taxane producing cells are transferred from the growth medium into a production medium that contains a higher concentration of the main carbon source. The production medium also includes a compound or complex containing silver and a compound containing methylene-dioxy. In addition, indanoyl amino acid compounds (eg, 6-EII), glutamine, and NAA (naphthalene acetic acid) are introduced into the cultures 0-3 days after the initial contact of the cells with the production medium. Even more preferably, these components are fed into the feed stream. Preferably, additional glucose or maltose is added to the culture as needed.

  Methods for purifying, recrystallizing, isolating, separating and extracting taxanes from cell cultures, plant tissues, or mixtures containing various taxanes are described in the literature (eg, US Pat. , Patent Literature 19, Patent Literature 20, Patent Literature 21, Patent Literature 22, Patent Literature 23, Patent Literature 24, Patent Literature 25, Patent Literature 26, Patent Literature 27, Patent Literature 28, Patent Literature 29, Patent Literature 30, Patent (Ref. 31, Patent 32, and Patent 33).

  When using the fed-batch method, it has been found that cells can be maintained in a highly productive state for a long period of time, and in fact, cell productivity could be enhanced. The supply configuration may be changed to achieve the desired result, such as increasing the production stage to increase taxane yield or increasing the growth stage to achieve higher biomass density. Will be apparent to those skilled in the art. Selection of conditions suitable to achieve optimal productivity and outcome is well within the ability of those skilled in the art in view of the teachings described herein. Similarly, other working parameters such as the timing and duration of addition and the rate of addition of fed-batch components to achieve the desired results can be varied in view of the teachings described herein. It is within the reach of the trader's ability.

  For example, as demonstrated in Bringi's US Pat. No. 6,057,834, removal of spent media and replenishment of fresh media on a regular basis, eg, every few days, significantly increased overall taxane and paclitaxel production, as well as cell This contributes to an increase in the amount of external products.

  The media exchange-promoting effect may be due to feedback inhibition and removal of in situ products that are thought to interfere with product degradation. Such favorable effects of in situ product removal on the production of secondary metabolites and secretion in irrelevant plant suspension cultures are particularly notable for Robins and Rhodes (1986) and Asada and Shuler (1989). Year). Periodic removal of spent medium includes the advantages described above, and further activates secondary biosynthesis by removing other non-taxane inhibitory components (such as phenolic compounds) from the medium. It can also be helpful.

  Supplementation of fresh media to cells undergoing active biosynthesis can also enhance production by providing deficient essential nutrients. For example, Miyasaka et al. (1986) was able to produce diterpene metabolites, cryptotanshinone, and ferruginol simply by stimulating tansine (Salvia miltiorhiza) stationary phase cells and adding sucrose to the medium. Perhaps biosynthesis stopped due to stationary carbon constraints. The regular media exchange protocol used in the present invention can be beneficial due to any of the above factors. Replenishment of depleted media components can also be achieved by a feed stream (feeding) containing those components.

  It is anticipated that the amount of medium replaced, the frequency of replacement, and the components of the medium supplemented may be varied according to various embodiments of the invention.

  The ability to promote biosynthesis and secretion through medium exchange has important implications for the design and operation of efficient continuous, semi-continuous, or fed-batch mass production processes.

  Any of the previously described embodiments for producing taxanes in culture may be used alone, but these techniques may be used in combination with each other.

  The following examples are non-limiting examples. It will be apparent to those skilled in the art that many changes and modifications can be made without departing from the spirit and scope of the invention described herein.

Example 1: using techniques generally recognized in the yew cell culture plant cell culture, starts Taxus cell cultures from any suitable part of the yew plant. On the solidified nutrient medium or liquid nutrient medium, substantially undifferentiated cells are grown under the conditions described below, except that the cells cultured in the solidified medium do not require stirring. Mix the culture by agitation at a temperature of 22-28 ° C., pH 4-7, in the dark, supply oxygen and other gases, aerate by contacting the oxygen-containing gas with the cell suspension, and Culture the cells. Oxygen is maintained between 10-150% of air saturation at working temperature and carbon dioxide is maintained at a concentration greater than 0.05%. Oxygen and other gas levels are controlled by adjusting agitation, pressure, gas composition, aeration rate, or gas supply rate. The medium contains components that can support the growth of yew cells, such as sugars in the range of 1-100 g / l, cumulative amounts of nitrogen source in the range of 1-100 mmol / l, and auxin and It may also comprise growth regulators such as cytokinin-like compounds, such as naphthalene acetic acid (NAA), phenoxyacetic acid, halogen-substituted phenoxyacetic acid, picloram, dicamba, benzylaminopurine, kinetin, zeatin, thidiazuron, indoleacetic acid. The medium may optionally be a taxane substance such as baccatin III or 10-deacetylbaccatin III, an amino acid such as glutamine, α- or β-phenylalanine, methylenedioxycinnamic acid (MDCA), or methylenedioxynitrosilicate. Cinnamic acid or methylenedioxyphenylacetic acid, or alpha-aminophenylacetic acid or related compounds, such as a silver ion source in the form of silver nitrate or silver thiosulfate, or other that can affect the biosynthesis or action of ethylene You may contain a component. The inoculum concentration of yew cells may range from 10 g fresh cell weight / liter to 300 g wet cell weight / liter.

  This medium also contains indanoylamide as described in this application, optionally with one or more jasmonic acid related substances, and these substances are also at the beginning of the culture, after the logarithmic growth phase, Alternatively, it may be added intermittently throughout the culture period. The other components of the medium may be added all at once or at various times during the culture and may be supplied continuously or intermittently. These components may be added either before or after plant cells are included in the culture broth. In addition, nutrients or other ingredients may be added during the culture period if deemed useful. Optionally, the medium may be changed after an appropriate culture period, whereby the cells are freshly exposed to a medium containing components similar to those described above. Such changes can include changing the amount of sugars such as glucose, fructose, sucrose, maltose, nitrates, ammonium, or nitrogen sources such as amino acids or casamino acids. After the incubation period, the culture is harvested, taxane levels are quantified by HPLC, and individual taxanes are identified using LC / MS / MS. Taxanes can be recovered from these cultures by appropriate extraction and purification procedures.

  In Examples 2-8, yew cells were cultured as callus cultures on solid media and then further cultured as a suspension of cell aggregates in liquid media. Although taxanes can also be produced by culturing in a solid medium, the use of a liquid medium is preferred. The culture temperature was adjusted between 20-30 ° C.

Example 2: Fed-batch addition of 6-EII This experiment was carried out according to the following conditions. The results of the 12-day fed-batch treatment are shown in Table 1, indicating that 6-EII and methyl jasmonate have similar activities. In accordance with the method described above, Chugokuichi cells were cultured as callus and then as suspended cells in a growth medium containing 1% maltose and B5 salt (medium A shown in Table B). After culturing in this medium for 7 days, the suspended cells are substantially separated from this medium, and about 20% (v / v) of fresh cells are obtained to be about 20% (w / v). Spent growth medium and 80% (v / v) basic production medium (Table B) with an initial pH of 5.8 and containing 20 μmol / liter MDCA and 50 μmol / liter SLTS (silver thiosulfate). Medium B shown in the above, but concentrated at 1.25 times). NAA (naphthalene acetic acid), GLN (glutamine), and MJS (methyl jasmonate), or 6-EII (6-ethyl-indanoyl-isoleucine) were fed as part of the feed stream at the following rate: 1.66 μmol / Liter / day (NAA), 0.84 mmol / liter / day (GLN), and MJS (2.51 μmol / liter / d) or 6-EII (0.832, 1.665, 4.17, or 6.25 μmol) / Liter / day). The gas phase was adjusted to 25% oxygen and 4.5% CO ₂ (the rest being nitrogen) and the temperature was maintained at 25 ± 2 ° C. Cells were cultured in the dark and stirred at 100-150 rpm for the duration of the experiment. After 12 days of culture in the production medium, the culture was harvested to obtain the taxane.

  The following results show that in cultures fed with 6-EII (ie, a cumulative dose of 20-100 μmol / liter) increment or methyl jasmonate (MJS), the production of taxanes was increased in methyl jasmonate. As well as to respond to 6-EII.

Example 3: Batch addition of 6-EII in combination with other media components Yew cells were cultured by a procedure similar to that used for Example 2. The medium consisted of various combinations of 6-EII (or methyl jasmonate-MJS), NAA, SLTS, MDCA, and glutamine. All ingredients were added batchwise, i.e. all added at once.

  The data showed that 6-EII and MJS have similar activities in improving taxane production. Therefore, 6-EII is probably effective even when used alone. However, the data also suggested some favorable interactions with media components. The data showed that 6-EII works synergistically with silver to increase the overall production of taxanes. In particular, these results demonstrate that 6-EII can improve baccatin III production without reducing paclitaxel production. Similarly, 6-EII and the auxin-type growth regulator NAA also interact positively and improve taxane production. The combination of 6-EII and an auxin type growth regulator is unexpectedly superior to using the individual components. The data showed that 6-EII and the amino acid glutamine interacted favorably to improve taxane production. Surprisingly, the data also showed that 6-EII, silver (supplied as SLTS), and the auxin-type growth regulator NAA interact to improve taxane production. Thus, with an appropriately prepared medium (such as medium B in Table B), NAA that is an auxin-type growth regulator, SLTS that is an example of a silver-containing compound / complex (an inhibitor of ethylene action), a methylenedioxy-containing compound ( Applying a combination of 6-EII with other potentiators such as phenylpropanoid metabolism inhibitors) and glutamine (an example of an amino acid) improves taxane production.

Example 4: Effect of 6-EII in combination with other media components This experiment was performed according to the following conditions. According to the method described above, Chugokuichi cells were cultured as callus and then as suspended cells on growth medium A (Table B). After culturing in this medium for 7 days, the suspended cells are substantially separated from this medium, and the basic pH is 5.8 so that the fresh cells are about 20% (w / v). Seeded in production medium (Medium B, Table B). In addition to the ingredients described in medium B, this medium further contained 5 mmol / liter glutamine. The gas phase was adjusted to 90-97% oxygen and 2.5-6% CO ₂ in air, and the temperature was maintained at 25 ± 2 ° C. Cells were cultured in the dark and stirred at 150-200 rpm for the duration of the experiment. After 14 days of culture in the production medium, the culture was harvested to obtain the taxane.

  Taxane yields as a function of specific component combinations in the media were tested and compared to the yields obtained in the absence of specific components or in the absence of specific component combinations.

  It was found that by including 6-EII in the range of 5-500 μmol / liter alone, the taxane yield was increased. However, when combined with 20 μmol / liter NAA (auxin-type growth regulator), the yield was improved significantly. The yield was further increased when 50 μmol / liter of silver (an inhibitor of ethylene action, supplied as a thiosulfate complex) was added to the combination of 6-EII and NAA. In addition, when 20 μmol / liter MDCA (3,4-methylenedioxycinnamic acid known to be an inhibitor of phenylpropanoid metabolism) was added to this combination, the yield was further increased. These surprising activities could not be predicted from the activity of the individual components. 20 μmol / liter NAA, or 20 μmol / liter NAA and 50 μmol / liter SLTS, or 20 μmol / liter NAA and 50 μmol / liter SLTS and 20 μmol / liter MDCA in combination with 50 to 200 μmol / liter, By adjusting under specific conditions, such as using an indanoyl amino acid such as 6-EII, it is also possible to optimize the concentrations of the components to maximize the desired result.

Example 5: Effect of Silver Nitrate and 6-EII on Taxane Production According to the method described above in Examples 1 and 2, Chugokuichi cells are then suspended as callus on growth medium (Medium A shown in Table B). Cultured as fresh cells. After culturing in this medium for 7 days, the suspended cells are substantially separated from this medium and medium B with an initial pH of 5.8 so that the fresh cells are about 20% (w / v). (Table B). In addition to the components listed in Table B, 20 μmol / liter NAA, 20 μmol / liter MDCA, 5 mmol / liter GLN, MJS or 6-EII, and silver as nitrate or thiosulfate complex at various levels in the medium Added to. The gas phase was adjusted to CO ₂ 90 to 97 percent oxygen and from 2.5 to 6% of the level in the atmosphere, the temperature was maintained at 25 ± 2 ° C.. Cells were cultured in the dark and stirred at 150-200 rpm for the duration of the experiment. After 14 days of culture in the production medium, the culture was harvested to obtain the taxane.

  The data demonstrated that 6-EII and silver act synergistically. That is, both 6-EII and silver, when used alone, do not produce the effect that is seen when cells are exposed to the presence of these agents in combination. In this example, silver nitrate and silver thiosulfate are used as illustrative examples of silver-containing compounds or complexes. The inventors have found that some other silver-containing compounds or complexes may be used. Examples of other suitable silver complexes can be found in disclosures such as, for example, US Pat.

Example 6: Effect of 6-bromoindanoylisoleucine (6-BII) on taxane production This experiment was performed according to the following conditions. According to the method described above, Chugoku ichiyi cells were cultured as callus and then as suspended cells on growth medium (Medium A, Table B). After culturing in this medium for 7 days, the suspended cells are substantially separated from this medium, and the basic pH is 5.8 so that the fresh cells are about 20% (w / v). Seeded in production medium (Medium B, Table B). In addition to the ingredients listed in Table B, the medium contained 20 μmol / liter NAA, 20 μmol / liter MDCA, 5 mmol / liter GLN, and 50 μmol / liter silver as a thiosulfate complex. 6-BII was added to the production medium at the levels shown in Table 2 below. The gas phase was adjusted to CO ₂ 90 to 97 percent oxygen and from 2.5 to 6% of the level in the atmosphere, the temperature was maintained at 25 ± 2 ° C.. Cells were cultured in the dark and stirred at 150-200 rpm for the duration of the experiment. After 14 days of culture in the production medium, the culture was harvested to obtain the taxane.

  The data indicate that, similar to 6-EII, 6-BII (halogen substituted derivative) was also an effective inducer of taxane production by Chugokuichi cell culture. The data also illustrates the determination of optimal 6-BII levels that could be determined by titrating other indanoylamides in taxane producing cell cultures.

Example 7: Effect of 1-oxo-indane-carboxy- (L) -isoleucine-methyl ester amide (1-OII) on total taxane accumulation by yew cell suspension culture This experiment was performed according to the following conditions. According to the method described above, Chugoku ichiyi cells were cultured as callus and then as suspended cells on growth medium (Medium A, Table B). After culturing in this medium for 7 days, the suspended cells are substantially separated from this medium, and the basic pH is 5.8 so that the fresh cells are about 20% (w / v). Seeded in production medium (Medium B, Table B). In addition to the ingredients listed in Table B, the medium also contained 20 μmol / liter NAA, 20 μmol / liter MDCA, 5 mmol / liter GLN, and 50 μmol / liter silver as a thiosulfate complex. 1-OII was added to the basic production medium at the levels shown in the table below. The gas phase was adjusted to 90-97% oxygen and 2.5-6% CO ₂ in air, and the temperature was maintained at 25 ± 2 ° C. Cells were cultured in the dark and stirred at 150-200 rpm for the duration of the experiment. After 14 days of culture in the production medium, the culture was harvested to obtain the taxane.

  The data show that 6-substituted derivatives, ie, indanoylisoleucine without substitution at the 6-position, as well as 6-EII and 6-BII described above, are also effective inducers of taxane production in yew suspension culture. It shows that there is.

Example 8: Effect of fed-feeding 6-EII and the effect of combining supplementation with additional sugar to the culture According to the method described above, in the growth medium A (Table B), Chugookuichi cells as callus, The cells were then cultured as suspended cells. After culturing in this medium for 7 days, the suspended cells are substantially separated from this medium, and an initial pH of 5.8 is obtained so that the fresh cells are about 20% (w / v). Seeded in medium B (Table B) concentrated 25 times, to which was added 20% (v / v) spent medium obtained from the end of culture in medium A. In addition, 20 μmol / liter MDCA and 50 μmol / liter silver as a thiosulfate complex were also added. NAA, GLN, and 6-EII were fed as part of the feed stream at the following rates: 1.66 μmol / liter / day (NAA), 0.84 mmol / liter / day (GLN), and 4 μmol / liter / day. (6-EII). The gas phase was adjusted to 25% oxygen and 4.5% CO ₂ (the rest being nitrogen) and the temperature was maintained at 25 ± 2 ° C. Cells were cultured in the dark and stirred at 100-150 rpm for the duration of the experiment. After a culture period under these conditions (12 days in this case), the culture was partially depleted of the main carbon source. At this time, the feed stream containing NAA, GLN, and 6-EII is supplemented with 400 g / l glucose so that an adequate supply of carbon source is provided to the cells and the duration of taxane production can be extended. did. In this way, the culture was continued for a further 16 days. At this point the culture was harvested to obtain the taxane and the amount of taxane was quantified using established analytical procedures.

  This result shows that, in contrast to batch culture where the effectiveness of the media components can limit the duration of taxane production, the supply of additional sugars in the fed-batch method allows this duration to be extended. Suggest to do. Furthermore, a large amount of taxane can be produced by supplementing sugar by adding a small amount by a fed-batch method instead of adding the whole amount at once.

  The foregoing describes preferred embodiments of the present invention along with some possible alternatives. However, these embodiments are merely examples, and the present invention is not limited thereto. Therefore, the results of the examples can generally be applied to yew species and are not limited to chum. Furthermore, the conditions of the examples such as the concentration of the added component are not limited.

Claims (29)

  A method for producing a taxane by culturing yew cells in a nutrient medium in suspension culture, wherein the indanoyl amino acid is added to the nutrient medium.   The method of claim 1, wherein the indanoyl amino acid is supplied in a fed-batch feed stream.   3. The method of claim 2, wherein the suspension medium is partially depleted of carbon source before additional carbon source is included in the feed stream.   The indanoyl amino acid is added to the nutrient medium in an amount effective to change the ratio between taxanes produced by the culture compared to the ratio obtained in the absence of indanoyl amino acid. Item 2. The method according to Item 1.   The indanoyl amino acid is added to the nutrient medium in an amount effective to selectively increase baccatin III compared to the amount obtained in the absence of indanoyl amino acid. the method of.   The method according to claim 1, wherein the indanoyl amino acid is a 6-position substituted indanoyl amino acid.   The indanoyl amino acids include 6-ethylindanoyl isoleucine (6-EII), 6-bromoindanoyl isoleucine (6-BII), and 1-oxo-indan-carboxy- (L) -isoleucine-methyl ester amide (1 The method of claim 1, wherein the method is selected from the group consisting of -OII).   The method according to claim 1, wherein the nutrient medium contains at least one enhancer and / or inhibitor.   The method according to claim 1, wherein the nutrient medium contains auxin, a compound having auxin-like growth-regulating activity, or both.   The method according to claim 1, wherein the nutrient medium contains silver ions, a silver compound, a silver complex, or a mixture thereof.   The method of claim 1, wherein the indanoyl amino acid is provided in a protective amount against silver toxicity.   The method according to claim 1, wherein the nutrient medium contains an inhibitor of phenylpropanoid metabolism.   The method according to claim 12, wherein the inhibitor is a compound having a methylenedioxy group.   The compound is 3,4-methylenedioxycinnamic acid, methylenedioxynitrocinnamic acid, methylenedioxyphenylpropionic acid, or 3,4-methylenedioxyphenylacetic acid. The method described in 1.   The method of claim 1, wherein the nutrient medium further comprises an amino acid.   The method according to claim 15, wherein the amino acid is glutamine.   A nutrient medium for plant cell culture comprising indanoyl amino acid.   The indanoyl amino acid is added to the medium in an amount effective to change the ratio between taxanes produced by cells cultured in the medium as compared to the ratio occurring in the absence of indanoyl amino acid. The medium according to claim 17.   18. The indanoyl amino acid is added to the medium in an amount effective to selectively increase baccatin III compared to the amount obtained in the absence of indanoyl amino acid. Culture medium.   The indanoyl amino acid includes 6-ethylindanoyl isoleucine (6-EII), 6-bromoindanoyl isoleucine (6-BII), 1-oxo-indan-carboxy- (L) -isoleucine-methyl ester amide (1- The medium according to claim 17, wherein the medium is selected from the group consisting of OII).   The medium according to claim 17, wherein the nutrient medium contains at least one enhancer and / or inhibitor.   The medium according to claim 17, wherein the medium contains auxin, a compound having auxin-like growth-regulating activity, or both.   The medium according to claim 17, wherein the medium contains silver ions, a silver compound, a silver complex, or a mixture thereof.   18. The medium of claim 17, wherein the indanoyl amino acid is provided in an amount that is protective against silver toxicity.   The medium according to claim 17, wherein the medium contains an inhibitor of phenylpropanoid metabolism.   The medium according to claim 25, wherein the inhibitor is a compound having a methylenedioxy group.   27. The compound is 3,4-methylenedioxycinnamic acid, methylenedioxynitrocinnamic acid, methylenedioxyphenylpropionic acid, or 3,4-methylenedioxyphenylacetic acid. The medium according to 1.   The medium according to claim 17, wherein the medium further comprises an amino acid.   The medium according to claim 28, wherein the amino acid is glutamine.